DNA Denaturation

FISH was carried out to study the mitotic chromosomes of root meristems. On the other hand, GISH was used to examine both the mitotic chromosomes of root meristemes and meiotic chromosomes of PMCs. Four probes were subjected to in situ hybridization on the same chromosome preparations. First FISH was made according to Książczyk et al. (2011 (link)) with minor modifications of Kwiatek et al. (2013 (link)), using 25S (used for detection of 25-5.8-18S rDNA loci) and 5S rDNA (pTa794). The hybridization mixture (40 μl per slide) contained 90 ng of each probe in the presence of salmon sperm DNA, 50 % formamide, 2 × SSC, 10 % dextran sulphate, and was denatured at 75 °C for 10 min and stored on ice for 10 min. Chromosomal DNA was denatured in the presence of the hybridization mixture at 75 °C for 5 min and allowed to hybridize overnight at 37 °C. For detection of the hybridization signals, anti-digoxigenin conjugated with FITC (Roche) was used. After documentation of the FISH sites, the slides were washed according to Heslop-Harrison (2000 (link)) (2 × 45 min in 4 × SSC Tween, 2 × 5 min in 2 × SSC, at room temperature).
Second FISH with pSc119.2 and pAs1 (labelled with digoxygenin-11-dUTP and tetramethyl-rhodamine-5-dUTP, respectively) was made with the same conditions after reprobing. After second reprobing, GISH was carried out according to Kwiatek et al. (2012 (link)) with modifications. Multicolour GISH was carried out using U-genome probe (from Ae. umbellulata), S^l-genome probe (from Ae. longissima) and unlabelled triticale genomic DNA which was used as specific blocker. The GISH mixture (40 μL per slide), containing 50 % formamide, 2 × SSC, 10 % dextran sulphate, 90 ng each of the genome probes, and 4.5 μg blocking DNA, was denatured at 75 °C for 10 min and stored on ice for 10 min. In case of initial GISH on triticale ‘Lamberto’ chromosomes, the hybridization mix contained the following: A-genome probe generated from genomic DNA of Triticum monococcum L., R-genome probe (rye, S. cereale L.) and blocking DNA from B-genome (Aegilops speltoides Tausch; 2n = 2x = 14; SS). The chromosomal DNA denaturation, hybridization and immunodetection conditions were the same as above-mentioned. Mitotic and meiotic (MI) cells were examined with an Olympus XM10 CCD camera attached to an Olympus BX 61 automatic epifluorescence microscope. Image processing was carried out using Olympus Cell-F (version 3.1; Olympus Soft Imaging Solutions GmbH: Münster, Germany) imaging software and PaintShop Pro X5 software (version 15.0.0.183; Corel Corporation, Ottawa, Canada). The identification of particular chromosomes were made by comparing the signal pattern of 5S rDNA, 25S rDNA, pSc119.2 and pAs1 probes according previous study (Kwiatek et al. 2013 (link)) and similar cytogenetic analysis (Cuadrado and Jouve 1994 (link); Schneider et al. 2003 (link), 2005 (link); Wiśniewska et al. 2013 (link)). Single-factor analysis of variance and Tukey’s Honest Significant Difference (HSD) test was used to examine the differences of means of chromosome configurations between plants from respective generations and the differences of means of chromosome configurations between plants from BC₂F₁ with comparison to their progeny in BC₂F₂ generation.

The jejunal mucosa was physically separated from seromuscular layers by scraping with a glass slide and was placed in RNAse free microtubes and immediately placed in liquid nitrogen. They were then stored at −80°C until use. Total RNA from jejunal tissue was extracted using the Qiagen RNeasy Minikit (Qiagen, Valencia, CA). Yield and quality of the extracts were determined by measuring absorbance at 260 and 280 nm (NanoDrop Technologies Thermo Fisher Scientific Wilmington, DE). The ratio of absorbance at 260∶280 was between 2.03 and 2.07. 1 µg of RNA was converted to cDNA using the iScript cDNA synthesis kit (Biorad) and pooled. The cycle conditions were 5 min at 25°C, cDNA synthesis at 42°C for 30 min, denaturation at 85°C for 5 min and held at 4°C. Primers were designed based on published sequences of the pig target genes either manually or using the NCBI online primer design tool (Primer-BLAST, http://www.ncbi.nlm.nih.gov/tools/primer-blast/), Primer3 input (version 0.4.0, frodo.wi.mit.edu/). The specificity of the primers was checked using the NCBI online Blast tool (Primer-BLAST, http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Quantitative RT-PCR was performed utilizing the iTaq Universal SYBR green Supermix (BioRad). Standard curves were generated using serial dilutions of pooled cDNA from all three normal pigs tested in triplicate. The StepOnePlus Real time PCR system (Applied Biosystems by Life Technologies, Carlsbad, CA) was used. Cycle parameters included polymerase activation and DNA denaturation at 95°C for 30 sec. Forty cycles of amplification were performed with a 15 sec denaturation at 95°C and annealing/extension and plate read for 60 sec at 60°C. The melting curve analysis was performed at 65°C–95°C at 0.5°C increments, 5 sec per step. Melting curves were checked to ensure consistent amplification of a single PCR product. Primer efficiency was calculated using the equation, Efficiency  = 10^∧(−1/slope) –1. All primer efficiencies were greater than 92%.

Using liquid nitrogen, fresh young leaves were ground to powder, and genomic DNA was extracted using the cetyltrimethylammonium bromide method (Fütterer et al., 1995 ). The forward primer for HMGR, FPS, DBR2, and HD1 was chosen from the EPSP marker gene. Because AaORA was driven by the CYP71AV1 promoter, the forward primer was designed within its sequence, and the reverse primer was selected from the CDS of AaORA.Table 1 shows a list of primers. The PCR reaction was carried out in a 20 μL tube using the LA TaqR Kit (Takara). DNA denaturation was set at 94° C for 3 minutes, followed by 30 cycles of 94° C for 30 seconds 57° C for 30 seconds, and 60 seconds at 72°C, and a final extension of five minutes at 72° C. For product determination, 1.0% agarose gel electrophoresis was performed.

Expression analysis was conducted by Real-Time Quantitative Reverse Transcription PCR (Real-Time qRT-PCR). The reaction mixture contained 100 ng of cDNA and the SYBR Green qPCR SuperMix-UDG (Cat. No. 11733046; Invitrogen, Carlsbad, CA, USA). PCR was performed using an iCycler IQ real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Specific primers for each selected gene are provided in Table S1. The amplification program included 35 cycles (30 s each) at 95 °C for DNA denaturation, followed by 62.3° or 57.6 °C for primer annealing and 72 °C for the extension. The PCR program included an initial 2 and 4 min periods at 50° and 95 °C, respectively, to activate the polymerase and a final 10 min extension at 72 °C. Alternative primer alignment temperatures corresponded to either target sequence. The expression of the 18S rRNA gene was followed in each sample as an internal reference, taking leaves to standardize comparisons among gene expression of seed development stages. The specificity of the PCR was assessed by the presence of a single peak in the dissociation curve performed after the amplification. Each quantitative PCR experiment was run three times separately and included three replicates in calculating the standard error for each sample. The results were analyzed by the 2-DDCT method (Livak and Schmittgen, 2001 (link)) with appropriate validation experiments.